Heart Disease is the leading cause of death in the United States and pathologies such as diabetes, hypertrophic cardiomyopathy, hypothyroidism and heart failure, as well as ischemia/reperfusion injury, involve alterations in myocardial contractile and regulatory proteins. The long range goal of our research is to understand the molecular mechanisms of contractile activation in cardiac and skeletal muscle. Contractile activation in both muscle types depends on 1) calcium binding to the thin filament and 2) myosin crossbridges that promote continued thin filament activation. However, evidence from this grant suggests the relative contribution of the two activation processes differs between skeletal and cardiac muscle, with skeletal muscle more dependent on calcium activation while cardiac muscle is more dependent on activation by strongly-bound crossbridges. These differences likely provide a mechanism for greater cellular level contractile control that is required for beat-to-beat regulation of cardiac output. Direct evidence to support this idea is difficult to obtain, in part due to an inability to alter crossbridge binding and calcium binding properties independently of one another. Our proposed experiments are specifically designed to separate calcium- and crossbridge-dependent components of activation. An additional strength of our research is that, under these conditions, we us a multiplicity of approaches to study the kinetic interactions between contractile and regulatory proteins in muscle cells. These include genetic engineering techniques, pharmaceutical interventions that alter the kinetics of Ca2+ binding to troponin C (TnC) or trap myosin in different crossbridge states, and use of mechanical transients and caged compounds to measure rates of chemo-mechanical steps in the CB cycle and isolated protein mechanical measurements. Additionally, x-ray diffraction will monitor myofilament structural changes and be coupled with mechanical measurements to determine how altered crossbridge kinetics influence activation processes. Specific aims of the proposal will investigate cardiac vs. skeletal differences in 1) the relative ability of strongly-bound cross-bridges to promote thin filament activation and force development kinetics; 2) the influence of calcium binding kinetics on thin filament activation and the rate force develops; 3) the dependence of activation processes on troponin isoforms; and 4) the dependence of activation on-tropomyosin isoforms. Results from this work will provide novel information regarding activation processes in cardiac muscle that allow fine-tuning of contractility at the cellular level in response to beat-to-beat alterations in venous return that can fail in cardiomyopathies.